The objectives of the prposed research are the determination of aspects of the structure of rabbit muscle phosphofructokinase and the elucidation of structure-function relationships for this important regulatory enzyme. The proposed approach will involve: a) cleavage of the phosphofructokinase protomer into its two independently-folded structural domains and an investigation of the relationships between these two structural domains and the regulatory and catalytic properties of the enzyme through kinetic and ligand-binding studies, b) determination of the locations of modifier and substrate binding sites and identification of the segments of the polypeptide chain that comprise these sites by affinity-labeling the sites, c) isolation, characterization and alignment of the set of peptides produced by cyanylation and cleavage of the enzyme at cysteine residues and the set of peptides generated by cleavage of the enzyme at methionine residues with CNBr, d) analysis of the amino acid sequence of the CNBr-generated peptides by the solid-phase Edman degradation method, and e) investigation of the molecular basis for regulation of phosphofructokinase through kinetic and conformational studies of the enzyme containing affinity labels covalently attached at regulatory sites.